PROJECT SUMMARY Staphylococcus aureus is a Gram-positive bacterium that causes devastating diseases, including skin and soft tissue infections, endocarditis, pneumonia, and sepsis. In order for bacterial pathogens to cause disease, small molecule cofactors and other important nutrients must either be synthesized or acquired from the host. S. aureus has the capacity to synthesize the iron containing cofactor heme as well as steal heme from host red blood cells. Unlike most cofactors, however, heme is toxic at high concentrations and bacterial pathogens experience heme toxicity during infection. S. aureus must balance the requirements for heme against its toxicity by regulating heme synthesis and heme acquisition. While the regulation of heme acquisition is well understood, very little is known regarding the regulation of heme biosynthesis in S. aureus or any other bacterial pathogen. Evidence from murine infections using mutant strains of S. aureus that are defective in heme synthesis implicates heme biosynthesis as necessary for colonization of the heart and liver, but not the bloodstream or kidneys. Thus, the bloodstream and kidneys are likely replete for host heme. The initial enzyme required for heme biosynthesis is HemA. Preliminary experiments have identified a membrane protein of unknown function, HemX, which regulates levels of HemA in a heme-dependent manner. Therefore, the central hypothesis of this proposal is that HemX regulates HemA to ensure heme synthesis in heme-deplete niches and reduce heme synthesis in host heme replete niches. We further predict that HemX is important for avoiding heme stress caused by excess heme synthesis in the bloodstream and kidneys. In Specific Aim 1, the regulation of HemA by HemX will be determined as transcriptional, translational, or post-translational. To identify genes other than HemX that are required to reduce HemA levels in heme-replete conditions, a transposon screen of a S. aureus HemA-YFP fusion strain will be performed. In combination with this genetic screen, proteins that interact with HemA and HemX will be identified by various cross-linking strategies and mass-spectrometry. These data will together define the mechanism of HemX regulation of heme synthesis. Experiments proposed in Specific Aim 2 will test the requirement of HemX for in vitro resistance to host oxidative and heme stresses. Next, using a murine infection model, experiments will test the contribution of HemX to colonization of various niches, determine the role of HemX in host-mediated heme stress during infection, and quantify abundance of HemA at different sites of infection. These findings have broad implications to understanding the role of heme synthesis regulation in the pathogenesis of bacteria that both synthesize and acquire heme during infection. Additionally, while virtually all cells make heme, the regulation of bacterial heme synthesis is poorly understood. This proposed work will contribute to our basic knowledge of regulatory mechanisms governing heme biosynthesis.